bioRxiv preprint doi: https://doi.org/10.1101/152082; this version posted June 20, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 1 Comparative analyses of venom-associated genes from an Old World 2 viper, Daboia russelii 3 1 1 4 Neeraja M Krishnan and Binay Panda * 5 1 6 Ganit Labs, Bio-IT Centre, Institute of Bioinformatics and Applied 7 Biotechnology, Biotech Park, Electronic City Phase I, Bangalore 560100 8 9 * Corresponding author: [email protected] 10 11 bioRxiv preprint doi: https://doi.org/10.1101/152082; this version posted June 20, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 12 Abstract 13 Molecular basis of toxin gene diversity among snakes is poorly 14 understood. Lack of whole genome sequence information for most snakes 15 makes studies on toxin genes and their orthologous counterparts difficult. One 16 of the challenges in studying snake genomes is the acquisition of biological 17 material from live animals, especially from the venomous ones. Additionally, in 18 certain geographies, Government permission is required to handle live snakes 19 making the process cumbersome and time-consuming. Here, we report 20 comparative sequence analyses of toxin genes from Russell’s viper 21 (Daboia russelii) using whole-genome sequencing data obtained from the skin 22 exuviate. In addition to the comparative analyses of 46 toxin-associated 23 proteins, we present evidence of unique sequence motifs in five key toxin- 24 associated protein domains; nerve growth factor (NGF), platelet derived 25 growth factor (PDGF), Kunitz/Bovine pancreatic trypsin inhibitor (Kunitz BPTI), 26 cysteine-rich secretory proteins, antigen 5, and pathogenesis-related 1 27 proteins (CAP) and cysteine-rich secretory protein (CRISP). We compared the 28 venom-associated domains from Russell’s viper with those from both 29 venomous and non-venomous vertebrates and invertebrates. The in 30 silico study on structures identified V11 and T35 in the NGF domain; F23 and 31 A29 in the PDGF domain; N69, K2 and A5 in the CAP domain; and Q17 in the 32 CRISP domain to be responsible for differences in the largest pockets across 33 the protein domain structures in New World vipers, Old World vipers and 34 elapids. Similarly, residues F10, Y11 and E20 appear to play an important role 35 in the protein structures across the kunitz protein domain of viperids and bioRxiv preprint doi: https://doi.org/10.1101/152082; this version posted June 20, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 36 elapids. Our study sheds light on the uniqueness of these key toxin- 37 associated proteins and their evolution in vipers. 38 39 Data deposition: Russell’s viper sequence data is deposited in the NCBI SRA 40 database under the accession number SRR5506741 and the GenBank 41 accession numbers for the individual venom-associated genes is provided in 42 Table S1. 43 44 Keywords: Toxin-associated genes, Russell’s viper, Old World vipers, New 45 World vipers, elapids 46 47 Introduction 48 Snake venom genes and their products offer an excellent model 49 system to study gene duplication, evolution of regulatory DNA sequences, 50 and biochemical diversity and novelty of venom proteins. Additionally, snake 51 venoms have tremendous potential in developing new drugs and bioactive 52 compounds (Vonk et al. 2011). Previous studies have highlighted the 53 importance of gene duplications and/or sub-functionalization (Malhotra et al. 54 2010; Rokyta et al. 2011; Hargreaves et al. 2014) and transcriptional/post- 55 transcriptional mechanisms (Casewell et al. 2014) contributing towards snake 56 venom diversity. Venom studies so far have extensively used peptides/protein 57 data alongside individual gene sequences or sequences of particular family 58 members to study variations on gene structure and sequence composition. Till 59 date, whole genome sequences of seven snake species, 60 king cobra Ophiophagus hannah (Vonk et al. 2013); Burmese bioRxiv preprint doi: https://doi.org/10.1101/152082; this version posted June 20, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 61 python Python bivitattus (Castoe et al. 2013); rattlesnake Crotalus 62 atrox (Dowell et al. 2016), Florida pygmy 63 rattlesnake Sistrurus miliarius barbouri (Vicoso et al. 2013); garter 64 snake Thamnophis elegans (Vicoso et al. 2013); five-pacer 65 viper Deinagkistrodon acutus (Yin et al. 2016); and corn 66 snake Pantherophis guttatus (Ullate-Agote et al. 2014), have either been 67 published or their sequence information available in the public domain. In 68 addition, genome-sequencing efforts are either underway or the sequences of 69 venom-associated genes have been deposited in the databases for few 70 others (Kerkkamp et al. 2016). Out of the sequenced genomes, only a few 71 have been annotated or the annotations have been made public, a key 72 requirement for comparative analysis of genes. This, along with the lack of 73 availability of whole genome sequences and/or complete coding sequences 74 for most snakes has made studies on toxin gene orthologies and gene 75 variation among venomous snakes limiting. 76 Four snakes, Russell's viper (Daboia russelii), saw-scaled viper 77 (Echis carinatus), spectacled cobra (Naja naja), and common krait 78 (Bungarus caeruleus) are responsible for most snakebite-related mortality in 79 India (Mohapatra et al. 2011; Whitaker 2015). Russell’s viper is a Old World 80 viper, member of the taxon Viperidae and subfamily Viperinae and is 81 responsible for large numbers of snakebite incidents and deaths in India. Very 82 little is known about the diversity of venom-associated genes from any viper, 83 including the only Old World viper where complete genome sequence 84 information is available (European adder, Vipera berus berus, 85 https://www.ncbi.nlm.nih.gov/bioproject/170536). Lack of any complete bioRxiv preprint doi: https://doi.org/10.1101/152082; this version posted June 20, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 86 genome annotation from this viper and other snake species reduces the 87 scope of a detailed comparative study on toxin-associated genes. Such a 88 study involving various groups of venomous and non-venomous snakes, in 89 addition to other venomous vertebrates and invertebrates, will facilitate our 90 understanding on the evolution of these genes, their diversity, and function. 91 One of the challenges in studying the genomes of venomous animals is 92 related to sample acquisition. Additionally, in India, Government permission is 93 required to catch snakes and extract blood samples from them (all snakes are 94 protected in India under the Indian Wildlife Protection Act, 1972). Even with 95 permission, there is a chance to adversely affect the animals during sample 96 acquisition. This may be circumvented by the use of skin exuviate (shed skin) 97 that does not require handling or drawing blood or taking any tissue from the 98 animals. However, working with DNA isolated from shed skin has its own 99 challenges. Microbial contamination, lack of full-length DNA in the exuviate 100 cells and computational challenges in dealing with short stretches of DNA are 101 some of the bottlenecks for working with DNA from exuviate skins. 102 In the current study, we explored the possibility of getting toxin gene 103 information from low-coverage whole-genome sequencing data using skin 104 exuviate from an Old World viper, Russell’s viper, and performed comparative 105 analysis on the annotated 51 venom-associated genes representing all the 106 major venom-associated protein families (Fry 2005) for which coding 107 sequences were available from a New World viper, a pit 108 viper, Protobothrops mucrosquamatus. We focused our analyses on five key 109 toxin-associated protein domains; nerve growth factor (NGF), platelet derived 110 growth factor (PDGF), Kunitz/Bovine pancreatic trypsin inhibitor (Kunitz BPTI), bioRxiv preprint doi: https://doi.org/10.1101/152082; this version posted June 20, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 111 cysteine-rich secretory proteins, antigen 5, and pathogenesis-related 1 112 proteins (CAP) and cysteine-rich secretory protein (CRISP) in Russell’s viper 113 and discovered key residues that are changed across Old World vipers, New 114 World vipers and elapids that might have contributed towards the evolution of 115 venom in vipers. 116 117 Materials and Methods 118 Russell’s viper skin exuviate and DNA isolation 119 Freshly shed skin exuviate of Russell’s viper from Bangalore, India was 120 a gift from Mr. Gerry Martin. The skin exuviate for the entire snake was 121 obtained, cleaned thoroughly with 70% ethanol and nuclease-free water 3 122 times each, dried thoroughly and frozen until the time of extraction of DNA.